Abstract: The Lung DNA Virome in Health and Disease We live in an interdependent relationship with our resident microbiota, which strongly influence human health and disease. Microbial communities differ at different body sites, and have distinctive consequences at each. The relationship between humans and their microbes has been most extensively studied for our bacterial communities but far less is known about the human virome, particularly in the respiratory tract. In preliminary studies, we used shotgun metagenomics to investigate the lung virome in several subject groups. We established efficient methods for purification of viral particles and computational methods for sequence assembly & taxonomic assignment. This revealed a diversity of phage & animal cell viruses. Most prominently, we discovered high levels in lung of Torque Tenoviruses (TTV), a family of small DNA viruses with extreme hypervariability, and we identified a novel group of small circular DNA viruses with limited homology to any previously described viruses (which we term ?human circo-related virus?; HCRV). It is known that TTV in blood is under potent host immune control, but neither cellular immune responses that control TTV in general nor lung-specific regulation have been investigated. Nothing is known about lung-specific HCRV since we identify them here for the first time. These findings highlight major gaps in current knowledge: How do the lung and its ubiquitous communities of viruses interact and affect each other, particularly the abundant small single stranded DNA viruses? What is the influence of cigarette smoking, a principal cause of lung injury? Are these viruses and host immune response to them contributing to lung injury and/or inflammation that drive lung diseases? To accelerate understanding of the lung virome and relationship to lung health & disease, we will: Phase 1 (R61) Years 1-2: To set the stage for further advancement, we will (1) Define the distribution of TTV & HCRV in respiratory tract samples from severely ill lung disease subjects (?extreme phenotype?); (2) Generate & interrogate lung virome metagenomic datasets for additional novel respiratory DNA viruses shared among individuals; (3) Develop a novel ?personalized? assessment of T cell immune responses to hypervariable TTV, and approach to HCRV immune responses; (4) Generate a repository of bronchoalveolar lavage (BAL) specimens representing lung diseases of high priority for virome & immune analysis in Phase 2. Phase 2 (R33) Years 3-5: We will (1) Define the relationship between lung virome constituents & virome- targeted immune responses, and pulmonary diseases of high priority; (2) Determine the impact of smoking on the lung virome and plasma virome, & virome-targeted immune responses; (3) Define the molecular virology of HCRV, TTV & other small DNA viruses relevant to the lung. This project is designed to be a combination of discovery/hypothesis-generating science as well as specific hypothesis-testing studies as outlined in RFA HL-17-002, which we anticipate will provide novel insight into the nature of the virome within the lungs, and role in lung health and disease.